Perspective Chapter: Applications of Biological Microlenses and Nanofibers

Abstract

In recent years, with the rapid development of micro/nano optics, biophotonics, and biomedicine, micro/nano optical devices have been widely used in biosensing, medical imaging, molecular diagnosis, and other fields due to their advantages of miniaturization and integration. However, micro/nano optical devices composed of semiconductor and precious metal materials are prone to irreversible physical damage to biological cells and tissues and require chemical synthesis, which cannot be naturally degraded in vivo. In addition, due to the limitation of solid materials, micro/nano optical devices are difficult to deform and move in practical applications such as optical imaging and signal detection. Therefore, it is necessary to find a natural, biocompatible, biodegradable, and controllable micro/nano optical device. During the evolution of nature, some organisms have formed bio-optical devices that can manipulate light beams. For example, algal cells have the ability to concentrate light, which can improve the efficiency of photosynthesis. Visual nerve cells have the ability to direct light and transmit images to the retina with low loss and distortion. These natural materials capable of light regulation bring new opportunities for biological micro/nano optical devices, which have potential applications in the assembly of biological cells, detection of biological signals, imaging in vivo, and single-cell diagnosis.

1. Introduction

High-sensitivity detection of biological signals and magnified imaging in vivo is of great significance in the diagnosis, prevention, and treatment of biological diseases. Among them, the application of micro/nano optical devices in the fields of life science research, environmental monitoring, and medical applications is growing, providing the possibility to detect and observe biological signals in complex environment [1]. At present, micro/nano optical devices based on surface plasmon resonance, optical micro/nanofibers, optical resonators, and photonic crystals have made significant progress in optical detection [2, 3, 4]. Surface plasmon-based optical detection uses metal film and medium surface to excite surface plasmon, and its resonant transmission changes with the change of medium refractive index. By observing the change of the characteristic parameters of the interaction between the incident wave and the surface plasmon, the change of the surface plasmon resonance transmission constant can be obtained [5]. With the continuous development of surface plasmon resonance, optical detection technology utilizing plasmon resonance of single metal nanoparticles or particle arrays can effectively improve the sensitivity and integrability of devices. When surface plasmons are confined in nanoparticles, and the size of particle is comparable to the incident wavelength, the free electrons of the particles can oscillate to generate localized surface plasmons [6]. The local surface plasmon resonance can be tuned by particle size, shape, and composition [7], but surface plasmon resonance is sensitive to interference factors such as sample composition and temperature. Unlike those based on surface plasmon resonance, optical micro/nanofibers are usually fabricated on thin films and low-refractive-index substrates using silicon or polymers [8]. When the light propagates in the micro/nanofiber film, the evanescent wave passes through the cladding covering the target molecule to sense the refractive index change of the sample, which can detect the optical signal [9]. In addition to optical detection using surface plasmon resonance and optical micro/nanofibers, optical detection can also be performed using optical resonators. When the incident beam is totally reflected in the cavity, the generated evanescent field can interact with the material on the surface of the resonant cavity, thereby realizing the detection of the signal in the cavity [10]. Most of the existing optical resonators are made of glass, semiconductor, and other materials [11]. Their surfaces can be functionalized, but the change of external factors will cause the shift of the resonant wavelength, thus affecting the performance of the optical resonator. Photonic crystals can be periodically arranged in space by two or more materials with different refractive indices, while introducing defects to break the problem that light cannot propagate in the crystal, providing new possibilities for controlling and manipulating light [12]. This structure has the advantages of low cost and integrability. These micro/nano optics have become powerful tools for applications such as optical imaging, signal detection, and disease diagnosis. With the rapid development of science, especially the advancement of biomedicine and nanophotonics, more and more studies have been conducted on micro/nano optical devices with high biocompatibility and good biodegradation for nano manipulation, optical imaging, and biosensing in a biological environment [13, 14, 15, 16]. The existing micro/nano optical devices are mainly composed of inorganic materials or metal materials. These materials have the advantages of high transparency and high refractive index, but they need to be chemically synthesized and cannot naturally degraded in vivo. Therefore, biological cells and tissues are susceptible to irreversible physical damage, thereby greatly limiting their applications in biological environments such as optical imaging and detection.

In the process of evolution of nature, some organisms have formed bio-optical devices that can regulate light beams, which provides inspiration for the construction of biocompatible and biodegradable micro/nano optical devices. These bio-optics are expected to realize biological signal detection, in vivo imaging, and disease diagnosis in biomedical applications. Based on the interaction between biological cells and light, biological microlens, biological optical micro/nanofiber, biological laser, and other micro/nano optical devices can be constructed [17, 18, 19]. Biolasers can be constructed through Fabry-Perot cavities and whispering gallery resonators to achieve cell labeling, diagnosis, and imaging. Bio-optical devices designed and assembled by biological cells or biological macromolecules have the advantages of biocompatibility, noninvasiveness, biodegradability, and resorbability, so they can perform optical detection and imaging in biological environments, offering great potential for bioimaging and therapy [20, 21].

This book chapter aims to review the fundamental principles and essential technologies of microlenses and nanofibers, along with their applications in biomedicine including optical devices, signal detection, and biological imaging. Specifically, it summarizes the fabrication of microlenses and nanofibers from natural biological materials like biological cells, spider silk, and diatoms. Furthermore, we have highlighted the issues and challenges faced by bio-inspired microlenses and nanofibers while exploring the future development directions of these technologies.

2. Biological microlens

2.1 Biological microlenses in nature

In nature, we are able to observe different types of microlens structures in animals and plants. In the optic nervous system, when a light beam enters the eye through the cornea, the refractive power of the cornea bends the light, so the light can pass freely through the pupil, and then, the light passes through the microsphere lens, where it converges into a focal point on the retina, and the image conversion is formed on the retina. The electrical impulses are transmitted by the optic nerve to the brain, as shown in Figure 1a. In addition, mitochondria in the eye’s photoreceptor cells also act as microlenses, helping transport light from the inside of the cell to the outside of the cell and converting it into neural signals. Ball et al. used confocal microscopy to observe the optical properties of mitochondria [22]. When the light from the light-emitting diode passes through the mitochondria, the side of the mitochondria can be focused into a beam with high brightness, and the half-width of the focused beam is 1 μm, as shown in Figure 1b. In plants, organisms such as algal cells convert solar energy into chemical energy through photosynthesis and store it in the plant. The stored chemical energy can be released to provide fuel for the activities of the organism and its life energy [26, 27]. Schuegers et al. discovered that cyanobacteria have phototaxis, which can directly and accurately perceive the location of light sources. This is because cyanobacterial cells act as microlenses, confining incident light to a focal point near the plasma membrane on the back of the light source. Photoreceptors on the cell membrane sense the light and drive the pili to pull towards the side close to the light source, thereby driving the cell to move towards the light source (as shown in Figure 1c) [23]. To measure the perturbations of light by cyanobacterial cells in the near field with high resolution, the team used a photolithographic method in which cyanobacterial cells were adsorbed on the surface of a photopolymer and UV light was projected vertically onto the cells so that each cell produces a sharp peak beneath the center. In addition, diatoms in plants also have the ability to focus light [24]. When the 100 μm red laser spot is reduced to 10 μm after passing through the diatom with regular geometrical structure, this focusing effect is produced by the superposition of the scattered waves from the holes on the surface of the diatom flap, which proves that the diatom has the function of microlens [28]. As shown in Figure 1d, as the illumination wavelength increases, the position of the focus of diatoms along the optical axis gradually decreases [25]. Researchers have gradually demonstrated that spherical or cylindrical objects with an index of refraction less than 2 can produce light-focusing effects.

Figure 1.

Biological microlenses in nature. (a) Schematic diagram of the eye focusing on light. (b) Vertical anatomy of a cone photoreceptor and optical properties of mitochondria under light [22]. (c) Schematic diagram of the near-field optical effects of cyanobacterial cells and cyanobacterial cells illuminated by light from different directions [23]. (d) Scanning electron micrographs and 3D renderings of diatoms [24] and the transmitted intensity of illumination light on the XY plane of diatoms [25].

Most living organisms are composed of proteins, water, and carbohydrates in nature. These biological materials have a refractive index of less than 2 under visible light conditions and can confine the light beam in the living organism, which makes the use of biological materials as Concentrators become possible [29, 30]. Monks et al. found that natural spider silk can act as a microlens [31]. When the spider silk is in close contact with the imaging object, the near-field evanescent waves of the imaging object can be transferred to the far-field by the spider silk, thereby realizing super-resolution imaging. This imaging process from near-field to far-field conversion is extremely sensitive to the gap distance between the spider silk and the object. Through experiments and simulations, it was found that when the gap between the spider silk and the imaging object is less than 100 nm, the micro/nano structure is magnified and imaged, thereby realizing super-resolution imaging. Therefore, under dry conditions, spider silk cannot achieve super-resolution imaging. Mammalian cells also exhibit lensing behavior. Miccio et al. demonstrated that suspended red blood cells can act as microlenses at the microscale. Due to the inherent elastic properties of erythrocytes, by changing the osmotic pressure, the shape of erythrocytes can be expanded from a disc shape to a spherical shape, and the focal length adjustment from negative to positive values is realized [32], thus demonstrating the imaging ability of erythrocytes and the advantages of adjustable focal length.

2.2 Application of biological microlenses in optical imaging

Synthetic microsphere lenses combined with optical microscopy imaging devices can be used in the field of biological imaging, but polymer microspheres are easy to cause damage to biological cells and tissues. When observing the structure of biological cells in vitro, it is necessary to stain the specific observation object, and then, the specific structure of biological cells can be observed through a fluorescence microscope [33]. However, biological cells, as a naturally occurring material, can focus light and guide light propagation in biological systems, acting as optical microlenses. Therefore, natural biomaterials with high biocompatibility and strong biodegradability have great potential in the field of bioimaging. In 2018, Liu et al. introduced a laser with a wavelength of 980 nm into a tapered fiber optic probe. The tip of the fiber optic probe was immersed in the red blood cell suspension. Under the action of the optical gradient force, the red blood cells could be stably trapped on the tip of the optical fiber. The structure can realize three-dimensional optical scanning imaging of single-cell membrane with a magnification of 1.7 times (as shown in Figure 2a) [34].

Figure 2.

Optical imaging of biological microlenses. (a) Schematic illustration of the assembled erythrocyte microlenses imaging the cell membrane [34]. (b) Using biological microlenses to image the intracellular cytoskeleton and two-layer structure on the cell membrane [35]. (c) Schematic diagram of the imaging of nanostructures by yeast cell chains [36].

Using the same method, the tip of the optical fiber can trap yeast under the action of light force, and the trapped yeast can be used as a biological amplifier, which can realize real-time magnification imaging of nanostructures and any position of biological samples under an optical microscope [35]. As shown in Figure 2b, under the ordinary light microscope, the intracellular fibrous cytoskeleton and the double-layered structure of the cell membrane are difficult to distinguish. When the yeast trapped by the optical fiber is placed above the epithelial cells, the incident light and the reflected light are passed through. The interference effect of the cytoskeleton can enhance the interaction between light and matter, and the double-layer structure of the cytoskeleton and the cell membrane can be clearly seen. This process does not require the use of specific fluorescent molecules to label cells, providing a direct imaging method for biomedicine. In order to increase the imaging field and imaging efficiency, Jiang et al. used fiber-optic tweezers to trap yeast [36]. Because of the spherical shape of the yeast, the capture laser can be focused to a very small area through the yeast and can exert a strong light force on the connected yeast, which are arranged in an orderly cell chain under the action of light superposition. When the cell chain is in the vicinity of the imaged sample, the biological chain can act as a near-field magnifying glass, capable of capturing sub-diffraction information of nanoscale objects under each cell and projecting them into the far field (as shown in Figure 2c). This label-free, real-time nanoimaging device lays a solid foundation for the development of super-resolution imaging devices and systems for biomedicine.

2.3 Application of biological microlenses in signal detection

Due to the advantages of high biocompatibility and good bioabsorbability of biological cells, biological cells have great potential in biosensing and signal detection. When a highly focused beam irradiates a biological microlens, the microlens can generate a subwavelength-sized focused spot in its near-field region, resulting in a high localized light intensity. This is due to the interaction between the scattering field of the microsphere lens and the incident light beam passing through the microsphere, which allows the microsphere lens to enhance the interaction between photons and substances under the illumination of the incident light, enhance the fluorescence signal, and realize the control of the object signal. Using biological microlenses to detect and enhance fluorescence signals provides a convenient and low-cost method for nanomaterial characterization and biomolecular diagnosis [37, 38, 39]. In 2017, Li et al. used spherical yeast and biological cells as natural biological microlenses to enhance upconversion fluorescence [40]. First, the optical fiber is placed in the upconversion nanoparticle suspension, and near-infrared light with a wavelength of 980 nm and an optical power of 3 mW is passed into the optical fiber probe. The upconversion nanoparticle suspension can be directly affected by the output laser of the optical fiber probe. As shown in Figure 3a and b when the optical force is used to trap biological cells at the tip of the fiber probe, the fluorescence intensity of the upconversion nanoparticle suspension excited by the incident laser is significantly enhanced. Under the action of the optical gradient force, the photon jet generated by the biological microlens can trap a single Escherichia coli and Staphylococcus aureus, which indicates that the existence of the biomicrolens can significantly enhance Escherichia coli and Staphylococcus aureus (as shown in Figure 3c–f). In addition, Staphylococcus aureus and Escherichia coli can be trapped and linked together under the action of light gradient force, and their upconverted fluorescence signals can be simultaneously enhanced by biological microlenses. In addition to yeast, mammalian cells can also be used as biological microlenses to enhance fluorescent signals. Since red blood cells do not have nucleus and organelles, they are disc-shaped with uniform refractive index distribution at pH 7.4. A single Escherichia coli can be trapped by red blood cells under the action of optical gradient force, and the fluorescence enhancement of single cells to nanoparticles can be realized. By lowering the pH of the erythrocyte solution, the morphology of the erythrocytes is swollen from the disk shape to a spherical shape under hypotonic conditions. When light passes through spherical red blood cells, a focused photon jet can be generated at the end of the red blood cells to excite strong fluorescence intensity of the upconverting nanoparticles modified on E. coli. Spherical human cells with larger diameters can also achieve enhanced fluorescence signals. For example, both K562 cells with a diameter of 10 μm and Raji cells with a diameter of 12 μm can enhance the upconversion fluorescence intensity of modified E. coli. This is because the surface of Raji cells and K562 cells is relatively rough and is a sphere with a nonuniform refractive index distribution. The experimental method can also be used to detect the capture process of single fluorescent polystyrene nanoparticles in real time [35].

Figure 3.

Optical detection of biological microlenses. (a and b) Biomicrolenses enhance the fluorescence of upconverting nanoparticles. (c–f) Enhanced fluorescence of single Escherichia coli and Staphylococcus aureus using biological microlenses [40].

Due to the diffraction limit of light, individual nanoparticles cannot be directly observed and detected under an optical microscope until the particles are trapped, although faint fluorescent spots from the nanoparticles can be seen in fluorescence mode. When a single nanoparticle was captured in the yeast’s focus, the nanoparticle was able to be seen clearly in both optical and fluorescence images due to the magnification mechanism of the microlens. When a single nanoparticle is released, the nanoparticle moves away from the focus of the biomagnifier due to the strong Brownian motion in the aqueous environment, and the size and fluorescence intensity of the nanoparticle observed under the microscope are compared with the case of the trapped nanoparticle somewhat reduced. In addition, since the highly focused beam generated by the microlens is illuminated on the nanoparticles, the backscattered signal of the trapped nanoparticles is enhanced. By analyzing the intensity and angular distribution of enhanced backscattering from nanoparticles located within the nanojet, one can derive the nanoparticle’s size and relative position with nanometer precision. In order to improve the sensitivity and biocompatibility of optical detection, Li et al. used yeast as a biological lens and used optical fiber probes to capture yeast to enhance the backscattering signal of E. coli chains [40], which provides applications for single-cell analysis and nanosensors.

3. Bio-optical micro/nanofiber

3.1 Bio-optical micro/nanofibers in nature

Signal detection and optical imaging of biological samples are of great significance in the research of biomedical applications such as biosensing and medical diagnosis. Because the biological optical micro/nanofiber can manipulate the light beam in a controlled manner, it will not cause irreversible physical damage to the biological sample during biological imaging and signal detection in the biological environment. Therefore, it is of great significance to construct biocompatible and movable biological optical micro/nanofibers. In the natural environment, natural optical micro/nanofibers can be widely observed in animals and plants. In the optic nervous system, the retina of vertebrates has specific optical properties, and light must penetrate multiple cell layers to reach the photoreceptor cells. As shown in Figure 4a, the geometry of Müller cells is similar to that of optical fibers so that Müller cells in their physiological environment can act as optical micro/nanofibers, allowing light to pass through the channels of the retina with low scattering. At the same time, the Müller cells arranged in parallel can direct light directly to their respective cone photoreceptors, thus maintaining the original image resolution and minimizing image distortion [41]. Furthermore, Müller cells enhance the signal-to-noise ratio by minimizing scattering and preserving the spatial distribution of light patterns in the propagating image [46]. Photons produced by bioluminescent organs are usually guided by some micro/nanofiber structure and emitted in a specific pattern. The jellyfish, for example, is mainly made of a translucent gel-like substance, the tunica media, which has a water content of up to 95–98%, and its bioluminescence is guided by fiber-like antennae to be used as bait to attract prey [47]; when the floating silkworm is stimulated externally, it can produce yellow light on its feet (as shown in Figure 4b and c) [42].

Figure 4.

Types of bio-optical micro/nanofibers. (a) Müller cells guide light from retina to photoreceptor cells [41]. (b) Optical microscope image of floating silkworm [42]. (c) Floating silkworms produce yellow light when stimulated [42]. (d) Light is directed through the stem of the plant to the roots [43]. (e) Fluorescence images of HY5 and protein fusions in wild-type seedling roots under light conditions [44]. (f) Optical image of a red laser guided by corn roots and oat seedlings [45].

In plants, some photoreceptors exist at the root of the plant, allowing the roots to sense different wavelengths of light [43]. As shown in Figure 4d, there are photoreceptors activated by light in root cells, which can act as natural optical micro/nanofiber devices to receive the stimulation of light beams. In addition, the roots of plants can receive information on light conditions through signal molecules and respond to the signal molecules, allowing the light beam to travel from the stem to the root. At the same time, the roots of the plant can also directly sense the light transmitted by the plant tissue. As shown in Figure 4e, the stem of the plant acts as an optical fiber that can conduct light to the phytochrome receptors in the root, triggering the production of the HY5 protein, which promotes healthy root growth [44]. As shown in Figure 4f, when the laser irradiated the corn roots and oat seedlings, the light energy was extended in a curve to the tips of the roots, which promoted the germination of the plants [45]. In addition, various optical micro/nanofibers can also be fabricated from naturally derived materials such as spider silk [48], cellulose [49], and bacterial cells [50] in nature. Spider silk has been shown to be an efficient optical fiber in various environments with an optical loss of 10.5 dB/cm. Natural spider silk fibers are also capable of delivering light in physiological fluids and integrated photonic chips. Through genetic engineering, large-scale production of spider silk proteins has become possible. Recently, researchers have created optical micro/nanofibers by using genetically engineered spider silk proteins [51]. In addition, the recombinant spider silk optical micro/nanofiber contributes to the efficient propagation of light due to its smoother surface, higher refractive index, and lower optical loss, which can deliver light to deep tissues with lower optical loss. Based on the light-guiding ability of a single cell, using optical power to assemble cellular micro/nanofibers can transmit light. Escherichia coli can be assembled into a biological micro/nanofiber using a tapered optical fiber by a laser with a wavelength of 980 nm. By changing the intensity of the laser light, the number of captured cells can be changed, thereby changing the length of the biophotonic micro/nanofiber.

3.2 Application of bio-optical micro/nanofiber in biomedicine

Natural biomaterials with high biocompatibility and biodegradability have been extensively studied for various medical applications such as drug delivery, biosensing, and optical imaging [52, 53, 54, 55]. Silk fibroin films were prepared from aqueous solutions of silk fibroin polymers, and crystallinity was induced and controlled by methanol treatment. In the process of processing into thin films, dextran and proteins of different molecular weights are encapsulated in the drug delivery device, and the drug release effect of silk fibroin can be evaluated by drug release kinetics [56]. In terms of biosensing and detection, light-capturing force is used to capture yeast cells and lactobacillus chains at the tip of an optical fiber. When light propagates through this biological chain, a beam with a half-width of 190 nm can be formed (as shown in Figure 5a) [57]. This biological micro/nanofiber can be used to detect the local fluorescence of leukemia cells in human blood; that is, when the biological micro/nanofiber is far away from the leukemia cells, no fluorescence can be detected. When the optical micro/nanofiber is in close contact with the cell membrane, a distinct fluorescent spot appears in leukemia cells stained with green fluorescent protein, which enables real-time detection of optical signals in the near field with subwavelength resolution. This biological cell-based optical micro/nanofiber can bend without damaging the cell membrane when it contacts the cell, and is highly flexible and deformable. At the same time, Wu et al. used the light-harvesting force to assemble a photofluidic cell chain of Enterococcus faecalis with a length of up to 360 μm and a propagation loss of 0.03 dB/μm [59]. When the incident light irradiates the cell chain, a beam with a focused beam waist radius of 400 nm can be obtained, which proves the micro/nanofiber performance of the E. faecalis cell chain. This biological micro/nanofiber can be used to detect backscattered signals from normal and pathological human red blood cells, providing a new approach for biocompatible devices for real-time biomedical sensing and single-cell diagnostics. In addition, the red blood cell micro/nanofiber can also be constructed using two tapered optical fibers. As shown in Figure 5b, red blood cells can be confined within the optical axis of two tapered optical fibers. By monitoring the light propagation of the red blood cell micro/nanofibers, the pH value of blood solutions can be detected in real time. This method can be used to diagnose pH-related issues. After diagnosis, optical torque is applied on the red blood cell micro/nanofiber, which rotates like a micromotor to transport the particles to the target area, and the red blood cell micro/nanofiber can be constructed in the zebrafish blood vessels (as shown in Figure 5c), providing a powerful tool for diagnosis and drug delivery [58]. In addition, spider silk as a natural optical micro/nanofiber enables the construction of biocompatible thermometers [60]. The spider silk used in the experiment was taken from the abdomen of the spider, and core-shell structured upconversion nanoparticles were assembled on the surface of the spider silk by photophoresis. Membrane temperatures of individual breast cancer cells were obtained by measuring the fluorescence spectra of upconverting nanoparticles on spider silk. This biomaterial-based thermometer enables real-time monitoring of temperature changes during apoptosis, providing a biocompatible tool for precise biosensing and single-cell analysis.

Figure 5.

Applications of bio-optical micro/nanofibers. (a) Bio-optical micro/nanofibers for single-cell detection [57]. (b) Schematic diagram of red blood cell optical micro/nanofiber for pH sensing [58]. (c) Schematic illustration of the assembly of red blood cell optical micro/nanofibers within zebrafish blood vessels [58].

4. Conclusions

To sum up, the noncontact and noninvasive signal detection and optical imaging of nanoparticles and biomolecules in the microenvironment has important research value and potential application prospects in the fields of biomedicine and nanophotonics. Biophotonic devices such as bio-microlenses and bio-optical micro/nanofibers have played an important role in scientific research. However, in the process of rapid scientific development, due to the good flexibility and membrane elasticity of biological materials, biological microlenses and biological optical micro/nanofibers have an important impact on the detection of microbial environment and the manipulation of living bodies. Using the radiation force generated by the focused optical field of the optical tweezers, operations such as capturing, stretching, and rotating the object can be achieved without contact and damage.

Acknowledgments

This work was supported by Guangdong Basic and Applied Basic Research Foundation (no. 2021B1515020046), National Key Research and Development Program of China (no. 2022YFA1206300), and National Natural Science Foundation of China (nos. 62135005, 62305132, and 12304322). Y.L. and H.L. contributed equally to this work.

Conflict of interest

The authors declare no competing financial interests.